1. Field of the Invention
This invention relates generally to genetic engineering, and more specifically, to gene therapy and to increasing viral stability.
2. Background Information
The goal of gene therapy is to treat disease by transferring genetic information into a diseased target cell. In comparison to conventional pharmaceutical therapy, gene therapy has greater potential for reduced treatment toxicity and for curing the disease. Gene therapy has been a major focus of biomedical research since the early 1980s when various methods were developed to introduce manufactured genes into cells. Gene therapies are under development for cancer, cardiovascular diseases, blood diseases, infectious diseases, neurologic disorders, inflammatory diseases and various genetic diseases. Approximately three million people each year in developed countries may benefit from gene therapy.
Methods to introduce exogenous genetic information into cells include physico-chemical as well as viral-mediated approaches. In general, viral-mediated approaches have received a great deal of attention because they can efficiently infect cells that express the appropriate viral receptors on the cell surface and because a wide variety of recombinant virus are available that differ in their host range and cell type infectivity. A wide variety of recombinant viruses have been successfully used to deliver exogenous genetic information to cells in vivo and in vitro. In particular, replication incompetent retroviruses have proved useful for gene therapy because they can infect most cell types and allow the stable introduction of genetic material into a chromosome of the infected target cell.
Retroviral infection of a cell, leading to stable integration of a viral nucleic acid molecule in the chromosome of the cell, is a process unique among RNA viruses. The process of retroviral infection begins with contact between the cell and the virus via specific receptor/ligand interactions. Following contact, the viral RNA is internalized and transcribed into DNA by the enzyme reverse transcriptase. The viral DNA then enters the nucleus and finally is integrated into the target cell's chromosomes. This last step requires cell division, which, for mammalian cells, occurs most efficiently at 37.degree. C.
The requirement for a target cell to undergo cell division for integration of the retroviral DNA limits the potential for high efficiency infection because only a small fraction of the cells in an unsynchronized population undergo cell division at any given time. Thus, high efficiency infection rates can be achieved only if active virus is present in a cell culture long enough for all of the cells to undergo cell division. Unfortunately, most recombinant retroviruses have a half-life at 37.degree. C. that is substantially less than the duration of one cell cycle. As a result, insufficient active virus is available for infection at the later rounds of cell division. This insufficiency could be prevented if high concentrations of retrovirus can be added to the cells in culture, or if retrovirus possessing a significantly-extended half-life can be used. A longer viral half-life reduces the need to concentrate the virus at later steps in processing.
Thus, there exists a need for methods to increase the stability of recombinant viruses so that a large number of target cells can be efficiently infected for gene therapy. The present invention satisfies this need and provides related advantages as well.